This invention relates generally to genetic engineering, and more specifically, to methods of increasing transfection efficiency of target cells.
Genetic engineering technology is used routinely to transfect cells. Transfection is the introduction of a foreign gene(s) into a target cell and the incorporation of that gene into a chromosome of the target cell. Once inside the target cell, a functional foreign gene can produce the RNA and protein product it encodes. Transfection has diverse applications in fermentation, research, agriculture, pharmaceuticals and medicine.
A particularly important application of transfection is gene therapy. Gene therapy has the potential to permanently treat diseases and deliver new therapeutic proteins and RNA that currently cannot be used. In gene therapy, a patient receives a functional foreign gene which produces a product that affects the disease or condition. Since the foreign gene can be stably incorporated into the patient's genome, the foreign gene has the potential to produce the product for the life of the patient. The patient receives the foreign gene by transfecting target cells ex vivo and administering the transfected cells to the patient or the foreign gene can be directly administered to the patient and the cells transfected in vivo.
A requirement of all transfection methods is that the foreign gene gets into the target cell. Many transfection methods have been developed but all can be classified as either direct or indirect methods. In direct methods, a genetic engineer injects the foreign gene into individual target cells using a microcapillary or microprojectile. Indirect methods involve the target cells passively or actively taking up the foreign gene. Indirect methods are diverse and include, for example, pinocytotic uptake of DNA-calcium phosphate and fusion of liposomes with the plasma membrane of the target cell. A very effective method is to use viral particles to infect the target cell because, once inside the target cell, foreign genes often express themselves at consistently higher levels by this method than by other methods.
Viral particles are themselves quite diverse and include DNA viruses, such as SV40, polyoma, adenovirus, Epstein-Barr, vaccinia, herpes simplex and baculovirus, and RNA viruses, such as tobacco mosaic virus, cucumber mosaic virus, brome mosaic virus and retrovirus. Retroviruses are particularly useful viral particles because, once inside the target cell, these viruses lead to stable transfections. Retroviruses which are replication-incompetent appear well suited for gene therapy because, in principle, these viruses do not produce any wild-type virus and cannot infect other cells after infecting the target cell. Replication-incompetent viruses are produced in so called "packaging cells" because these cells "package" the foreign gene into viral particles which can infect, but not replicate.
The major problem with indirect transfection methods is that they are inefficient at transfecting target cells. Transfection efficiencies of 1-20% are achieved but, for human target cells, the transfection efficiency is at the lower end of the range. The transfection efficiency is the number of target cells containing at least one copy of the foreign gene divided by the total number of target cells. Thus, most current indirect transfection methods waste large amounts of costly target cells, carriers and foreign genes because only a small fraction of exposed target cells is transfected. This inefficient method particularly limits development of gene therapy because gene therapy requires many transfected target cells. In certain circumstances, higher transfection efficiencies are possible but often heroic measures are needed to achieve them. For example, bone marrow target cells can be cultured for several weeks with repeated exposures to retroviral particles. Such methods are not practical because of expense, complexity or incompatibility of the target cells and particles. There is a need for a more efficient, easy-to-use, generally applicable transfection method.
Contact between target cells and viral particles is essential for transfection to occur. Generally, indirect viral transfection occurs by culturing target cells with viral particles suspended in the cell culture medium. All indirect transfection methods are based on random contact between viral particles and target cells. Typically, the culture is gently agitated during transfection and suspended viral particles contact target cells by chance. Although specific target cells can be selected for transfection using various techniques, the contact between target cells and viral particles in these methods remains a random event. Methods for selectively transfecting target cells include bridging antibodies between viral and target cell antigens and chemically modifying particles for specific target cell receptors. Current indirect transfection methods are therefore limited to random contact between viral particles and target cells.
Increasing the concentration of viral particles increases contact between viral particles and target cells. However, the viral particle concentration that can be used for transfection is limited because the proportion of infectious viral particles decrease as the viral particles are concentrated. Various methods are used to concentrate retroviral particles including polyethylene glycol precipitation, sucrose gradient centrifugation, pelleting by centrifugation, aqueous two-phase systems, ammonium sulfate precipitation, and hollow fiber ultrafiltration. A measure of viral particle concentration is titer that, for replication-incompetent retrovirus, is typically about 10.sup.4 to 10.sup.5 colony forming units (CFU)/ml. Viral particle concentration limits the transfection efficiency of current viral transfection methods by limiting the contact between viral particles and target cells.
Current indirect transfection methods require chemical additives to transfect target cells. Chemical additives allow viral particles to enter target cells more easily. Chemical additives include, for example, polybrene and protamine sulfate. In current methods, chemical additives are required because particle-target cell contact is so infrequent it is necessary to maximize the number of particles that enter target cells once contact occurs. Without chemical additives, even the relatively low transfection efficiencies achieved by current methods would not be possible. Chemical additives are undesirable for gene therapy because the chemical additives pose a contamination concern.
Another problem with current methods is that some target cells cannot be transfected because the particles cannot contact the target cells in culture. For example, hematopoietic stem cells (HSCs), a prime target for gene therapy applications, are often grown in cell culture in association with accessory cells (stromal cells). The HSCs position themselves between stromal cells and the cell growth support, and become physically inaccessible to retroviruses in the cell culture medium. HSCs cannot be transfected by retroviruses because the stromal cells block retroviral access to the HSC. A method is needed which allows particles to contact target cells even though the target cells are covered over by accessory cells.
Besides contact limitations, low transfection efficiencies can result from cell culture inhibitors that limit target cell growth. Retroviruses require dividing target cells to transfect. Packaging cell culture supernatant contains growth inhibitors that reduce target cell growth. Since the target cells must divide for transfection to occur, inhibitors reducing target cell growth reduce transfection efficiencies. Using current methods, it is difficult to remove inhibitors in the packaging cell culture supernatant from the replication-incompetent retroviruses.
Clearly, there is a need for new transfection methods that improve the efficiency of target cell transfection. New transfection methods are needed which increase particle-target cell contact without adversely effecting particle infectivity and do not require chemical additives to transfect target cells. Further, new transfection methods are needed to transfect target cells that are not normally accessible in culture and to remove growth inhibitors from transfecting cultures. These needs are particularly acute in the field of gene therapy. The present invention satisfies these needs and provides related advantages as well.